Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V006523 | pRK5-HA-Ubiquitin-K48 | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pRK5-HA-Ubiquitin-K48
- Antibiotic Resistance:
- Ampicillin
- Length:
- 4996 bp
- Type:
- Mammalian Expression
- Replication origin:
- ori
- Copy Number:
- High Copy
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- SP6
pRK5-HA-Ubiquitin-K48 vector Map
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
pRK5-HA-Ubiquitin-K48 vector Sequence
LOCUS 40924_37158 4996 bp DNA circular SYN 02-SEP-2021
DEFINITION Mammalian expression of HA tagged ubiquitin with only K48, other
lysines mutated to arginines.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4996)
AUTHORS Lim KL, Chew KC, Tan JM, Wang C, Chung KK, Zhang Y, Tanaka Y, Smith
W, Engelender S, Ross CA, Dawson VL, Dawson TM
TITLE Parkin mediates nonclassical, proteasomal-independent ubiquitination
of synphilin-1: implications for Lewy body formation.
JOURNAL J Neurosci. 2005 Feb 23. 25(8):2002-9.
PUBMED 15728840
REFERENCE 2 (bases 1 to 4996)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 4996)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J Neurosci.
2005 Feb 23. 25(8):2002-9."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4996
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 14..393
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 394..597
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 594..618
/label=LNCX
/note="Human CMV promoter, forward primer"
promoter 828..846
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 859..878
/label=pMT2-F
/note="Synthetic intron, forward primer"
regulatory 963..972
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 975..1001
/codon_start=1
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
/translation="YPYDVPDYA"
CDS 1032..1259
/codon_start=1
/label=ubiquitin-K48
/note="human ubiquitin variant containing Lys-48, with all
other lysines mutated to arginine"
/translation="MQIFVRTLTGRTITLEVEPSDTIENVRARIQDREGIPPDQQRLIF
AGKQLEDGRTLSDYNIQRESTLHLVLRLRGG"
regulatory 1296..1305
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
polyA_signal 1309..1443
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
promoter 1512..1869
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(1889..1905)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 2118..2573
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2590..2609)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 2709..2731
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(2769..2787)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 2855..2959
/label=AmpR promoter
CDS 2960..3817
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 3991..4579
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4733..4750
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 4867..4888
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 4903..4933
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4941..4957
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
primer_bind 4965..4981
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"